亚甲基四氢叶酸还原酶C677T和内皮胞蛋白C受体A6936G基因多态性与深静脉血栓形成的相关性研究
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摘要
研究背景：
深静脉血栓形成(DVT)是指血液在深静脉内不正常凝结,阻塞管腔,导致静脉回流障碍的一种疾病。深静脉血栓形成的主要症状为肢体肿胀和疼痛,症状随着血栓的蔓延可能会逐渐加重,少数严重病例可以发生股青肿甚至静脉性坏疽。而且如果不能及时妥当地诊断和处理,有些症状会长期迁延,最终会演变为静脉血栓形成后综合征,长期影响患者的生活质量；还有一些病人可能会因血栓脱落而形成肺栓塞,直接危及病人生命,或者演变为慢性栓塞性肺动脉高压。目前,我国DVT的发病率尚无确切的统计资料,据国外资料报道,美国DVT年发病率约为千分之一。
个半世纪以前,Rudolph Virchow提出了血液瘀滞,血管壁的变化,和血液高凝状态是机体发生发展静脉血栓的主要机制,这个理论现今仍然适用,基本上所有的导致血栓的因素,都影响这三种机制之一。目前认为有很多导致个体易患静脉血栓的因素,可分为两类：一种是环境及后天生理病理因素的改变,如外伤,妊娠,围手术期,恶性肿瘤,机体制动,药物等影响机体的凝血机制；另一种是由遗传基因突变参与的凝血机制异常,有研究认为遗传基因突变在大于30%的个体参与到了深静脉血栓的发生过程。人们对各种基因多态性与血栓形成之间的关联性做了相当深入的研究,也发现了越来越多的基因多态现象确实参与到了凝血机制的改变和调节,并与静脉血栓构成相相关性。目前有研究认为亚甲基四氢叶酸还原酶C677T基因多态性由于会导致亚甲基四氢叶酸还原酶活性下降,进而影响同型半胱氨酸代谢,形成高同型半胱氨酸血症,从而促进血液高凝,增加机体血栓形成的风险；内皮细胞蛋白C受体A6936G基因多态性由于会提高血浆sEPCR的水平<进而增大SEPCR对蛋白C抗凝途径的负向调节,从而形血液高高凝,增加机体血栓形成的风险。对这两类基因多态现象的研究已经成为热点,本课题以临床病例为基础,采用一定的试验方法,对亚甲基四氢叶酸还原酶C677T基因多态性和因皮细胞蛋白C受体A6936G基因多态性与深静脉血栓形成的相关性进行了研究。
第一部分亚甲基四氢叶酸还原酶C677T基因多态性与深静脉血栓形成的相关性研究
亚里基四氢叶酸还原酶(MTHFR)在同型半胱氨酸(Hey)代谢中起着关键作用,可以催化5,10-亚甲基四氢叶酸转换为5甲基四氢叶酸,从而成为甲基的提供者参与维生素B12依赖的同型半胱氨酸向蛋氨酸转化；MTHFR降低,会导致Hey的代谢障碍,进而形高同型半胱氨酸血症。最近的研究已经证实高血浆Hey浓度是在深静脉血栓形成的勺危险因素之一，而MTHFR(C677T基因多态性已被报道可能会产生MTHFR酶的热不稳定性以及一定程度上降低该酶的活性,进而形成高同型半胱氨酸血症,从而导致机体易形成血栓。为了进一步深入研究探讨这一理论,我们设计了本项病例对照研究。
目的与方法
探讨血Hey水平及MTHFR C677T基因多态性与DVT发病的关系。在本课题的病例对照研究中,我们分析了DVT患者的MTHFR基因C677T多态性各基因型的频率，并与正常对照进行比较。此外,通过检测受试者血浆Hey水平，我们评估这一多态性与血浆Hey水平之间的关联,以及血浆Hey水平与DVT的关系。应用酶联关免疫吸附试验(ELISA)检测血浆Hey水平，采用限制旧性片段长度多态性聚合酶链反应(PCR RFLP)技术测定基因组DNA。
结果
MTHFR基因在677位置菜有3种基因型CC，CT和TT基因型，T为突变等位基因。在DVT组,T等位基因频率明显高于对照组。与CC基因型相比,TT基因型与DVT相关的风险显著增加(OR=3.200；95%CI,1.2937.922，P=0.010。与等位基因(相比,突变等位基因T与DVT的风险增加相关(OR=1.712;95%CI,1.095-2.675,P=0.018),DVT组的血浆Hey水平显著高于对照组而且MTHFR基因C677T突变对于血浆Hey水平有影响,突变等位基因T与血浆Hey水平升高有关。
结论
MTHFR677位点基因突变与个体发生DVT的风险增加之间存在关联。突变纯合子可以导致DVT、风险增加,突变等位基因T也与DVT风险增加有关。DVT患者有着比健康人群更高的血浆同型半胱氨酸水平,而且MTHFR基因C677T突变对于血浆Hcy水平有影响,突变等位基因T与血浆Hey水平升高有关。结论是,MTHFR C677T基因多态性可能是DVT发生的危险因素之一,而这种关联可能是通过血浆Hcy的浓度变化实现的。
第二部分内皮细胞蛋白C受体A6936G基因多态性与深静脉血栓形成的相关性研究
已知蛋白C (PC)系统是一种重要的抗凝血机制,在其辅酶蛋白S(PS)存在的条件下,活化蛋白C (aPC)使因子Va和Villa失活,从而减少凝血酶生成。内皮细胞蛋白C受体(EPCR),是一种在血管内皮细胞的表面的受体。这种受体可以与PC或aPC结合。这种结合可以提高PC的活化效率,加速aPC对因子Va和Villa灭活,从而实现抗凝。而可溶性内皮细胞蛋白C受体(sEPCR)在金属蛋白酶作用下从EPCR脱落,也可以与PC和aPC结合,但它与EPCR作用相反,而且产生了与EPCR的竞争性抑制,从而抑制了aPC的抗凝活性,导致高凝。因此血浆sEPCR水平被认为与机体的高凝倾向有关,sEPCR水平升高会导致机体易患血栓,而EPCR基因突变可能会产生sEPCR水平的升高。为了进一步探讨这个理论,我们对EPCR A6936G多态性、血浆sEPCR水平和DVT之间的关系进行了深入的研究。
目的与方法
本研究通过检测DVT病人以及正常对照人群EPCR A6936G多态性分布,并通过测定血浆sEPCR表达水平,探讨其在DVT形成中的作用。病例组由已确诊的DVT患者组成,对照组来自健康查体者。应用酶联免疫吸附试验(ELISA)检测血.浆sEPCR水平,采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术测定基因组DNA。
结果
DVT组突变等位基因G的频率显著高于对照组。相对于AA基因型,AG基因型(OR=2.502;95%CI,1.215-5.153),和等位基因G携带者AG+GG(OR=2.507;95%CI,1.238-5.076),都表现出增加了DVT的发生风险。突变等位基因G相对于等位基因A,也增加了DVT发生的风险(OR=2.178；95%C1,1.159-4.093)。DVT组血浆sEPCR水平显著高于对昭组,而突变等位基因G与血浆sEPCR水平升高有关。
结论
EPCR6936的基因突变与机体DVT发病的敏感性有关,突变等位基因G导致DVT发病危险增加。DVT患者具有比健康群体史更高的血浆sEPCR水平,而且血浆sEPCR水平与EPCR A6936G基因多态性存在关联,基因突变会导致血浆sEPCR水平升高。结论是,EPCR A6936G的基因多态性与血浆sEPCR水平升高有关,其可能是人群中发生DVT的危险因素之一。
课题创新性与意义
一.本研究结合了流行病学的研究方法和分子生物学的实验技术,首次同期联合检测了DVT患者和健康人群的MTHFR基因C677T和EPCR基因A6936G多态性的分布以及与其关联的功能蛋白Hey和sEPCR的表达水平,确定两个基因多态性与DVT之间的联系,以及基因多态性与功能蛋白表达水平的关系。为从病因学上在基因水平研究血栓性疾病增加了新的认识。
二.本课题体现了“基因-蛋白-疾病”的研究思路,由于本研究对象集中于特定的地理区域(山东),实验结果具有一定的地域代表性,同时实验结论也具有临床指导意义；此外,本研究从方法学上为进一步研究基因多态性提供了一定依据。
Backgrounds
Deep venous thrombosis (DVT) in medicine is a serious and common disease affecting all age groups. Its causes include a variety of pathological conditions and it often lead to hospitalization of patients, and severe conditions that require urgent intervention. About one and a half century ago, Rudolph Virchow proposed the main determinants that lead to DVT, summarizing that blood stasis, changes in the vessel wall, and blood hypercoagulability are the main mechanisms of the body for developing venous thrombosis. This theory is still applicable today and almost all of factors that lead to thrombosis, either systemie or of molecular level, have essentially affected one of the three mechanisms.There are a lot of mechanisms lead to the susceptibility to venous thrombosis clinically.which can be divided into two main categories:one category includes direct or indirect causes by trauma, systemic disease, or physiological state changes such as pregnancy, high-altitude, surgery, malignancy, body immobilization, and so on. The other is due to genetic mutations involving in the abnormal blood clotting mechanism. At present, due to the rapid progress of molecular genetics researches, and more accurate in vitro experiments, it has been found that in more than30%of individuals, genetic mutations participate in the mechanism of occurrence of DVT. Domestic research in this area is relatively rare. The present study is divided into two parts, respectively, related to endothelial protein C receptor A6936G gene polymorphism and methylenetetrahydrofolate reductase C677T gene polymorphism. Moreover, the relationship between the two polymorphisms and DVT were also studied.
Part1Deep Venous Thrombosis has Relationship with Methylenetetrahydropholate Reductase C677T Gene Polymorphism and Hyperhomocysteinemia
Methylenetetrahydrofolate reductase (MTHFR) plays a key role in homocysteine (Hey) metabolism,which can catalyze the reaction from5,10-methylenetetrahydrofolate to5-methylenetetrahydrofolate, thus becoming the methyl donor involved in the conversion from vitamin B12-dependent homocysteine to methionine. Reducing MTHFR concentration,it tend to be observed that thermal instability form of the enzyme, it will lead to high homocysteine concentration, which is characterized by mild to moderate increasing in plasma total homocysteine concentration. In vascular occlusive disease, the relationship with hyperhomocysteinemia has been reported. Recent studies have also demonstrated that high plasma total homocysteine concentration is a risk factor for deep venous thrombosis (DVT) in the general population.
MTHFR gene polymorphisms have been reported to be associated with high plasma homocysteine concentration. MTHFR gene C677T,can develop thermal instability and reduce the enzyme activity in vitro to a certain extent. C677T mutation in the homozygous individuals can moderately increase plasma homocysteine concentration, especially under the condition of low plasma folate level. Homozygotes of the C677T polymorphism in some studies have been regarded as a risk factor for venous thrombosis, however, not all studies support this conclusion. Opposite conclusions exist in some researches.Moreover, few domestic research has reported in this area.
Objective and Methods
To explore the relationship of blood I Icy levels and MTHFR C677T gene polymorphism and DVT incidence, in this case-control study, we analyzed the frequency of genotypes with MTHFR C677T polymorphism in DVT patients compared with normal individuals without DVT. In addition, we evaluated the association between this polymorphism and plasma Hey levels. PCR-RFLP was used to investigate the relationship between MTHFR C677T genotypes and DVT. The Hey levels were measured by enzyme-linked immunosorbent assay (ELISA) in DVT patients and control subjects.
Results
There are3genotypes,CC, CT and TT genotypes,in the MTHFR gene at position677in the two groups. The frequencies of T allele in DVT group was significantly higher than in the controls. The MTHFR C677T frequencies TT genotype was also significantly higher than in the controls. Compared with the CC genotype, the TT genotype was significantly correlated with a increased risk of DVT (OR=3.200;95%CI,1.293-7.922, P=0.010). Compared with the C allel, the T allel was significantly correlated with a increased risk of DVT(OR=1.712;95%CI,1.095-2.675, P=0.018).The plasma level of I Icy in the DVT group was significantly higher than that in the control group. And the level of Hcy in subjects with TT genotype was significantly higher than in subjects with CT genotype, meanwhile, the level of Hcy in subjects with CT genotype was significantly higher than in subjects with CC genotype.
Conclusion
The analysis in this study showed that the MTHFR677TT homozygote was a significant risk factor for DVT. And the I allel was correlated with a increased risk of DVT. There are differences in I Icy levels among the different genotypes impling that the mutation accounts for the changes of Hey levels. Hcy levels are elevated in DVT. In conclusion, our present observations allow us to suggest that the C677T mutation of the MTHFR gene, which is possible to cause hyperhomocysteinemia, is probably one of the candidate genetic risk factors of DVT.
Part2Endothelial cell protein C receptor gene6936A/G polymorphism is associated with deep venous thrombosis
Protein C (PC) system is an important natural anticoagulant mechanism. Under the condition of the existance of its coenzyme protein S, activated protein C (aPC) can make inactivation of factor Va and Ⅷa, thereby reducing thrombin generation. The activation of endothelial cell protein C receptor (EPCR), is a vascular endothelial cell surface receptor of newly discovered. This receptor combining with almost the same affinity with PC or aPC,can accelerate the activation of PC.Whereas, soluble endothelial cell protein C receptor (sEPCR) gets the effect of metalloproteinase, shedding from EPCR, being also combined with PC and aPC similar affinity. sEPCR is able to block the aPC with the combination of phospholipids and eliminate its effects of inactivation for factor Va, thereby inhibiting the anticoagulant activity of aPC. Several factors can influence level of sEPCR including gene polymorphisms. For further investigating the relation between EPCR A6936G polymorphism and DVT,we performed this case-control study. The relation between EPCR A6936G polymorphism and sEPCR level was also studied.
Objective and Methods
In this study, the aim of this study was to explore the role of gene mutation in the DVT formation. PCR-RFLP was used to investigate the relationship between EPCR A6936G polymorphism and DVT. The plasma soluble endothelial cell protein C receptor (sEPCR) levels were measured by enzyme-linked immunosorbent assay (ELISA) method in DVT patients and control subjects. All statistical analysis was performed for confirmation the possible relationships.
Results
Three genotypes, AA, AG and GG, in the EPCR gene at position6936were noted in the DVT group as well as in the control group. In the DVT group, the frequency of mutational genotypes was significantly higher than that in the control group (P<0.05). Furthermore, the frequency of the G allele in the DVT group was significantly higher than that in the control group (OR=2.178;95%CI,1.159-4.093). The plasma level of sEPCR in the DVT group was significantly higher than that in the control group. And the level of sEPCR in subjects with GG genotype was significantly higher than in subjects with AG genotype, meanwhile, the level of Hey in subjects with AG genotype was significantly higher than in subjects with AA genotype.
Conclusion
In this study, the results suggest an increased risk for venous thrombosis in EPCR6936mutational genotypes. The G allele indicates susceptibility to DVT and is associated with DVT pathogenesis. Moreover, our results indicate that the plasma sEPCR level is associated with the A6936G polymorphism of the EPCR gene. The plasma sEPCR level was elevated in DVT patients. Our findings suggest that the EPCR gene A6936G polymorphism may be a candidate risk factor for DVT.
深静脉血栓形成(DVT)是指血液在深静脉内不正常凝结,阻塞管腔,导致静脉回流障碍的一种疾病。深静脉血栓形成的主要症状为肢体肿胀和疼痛,症状随着血栓的蔓延可能会逐渐加重,少数严重病例可以发生股青肿甚至静脉性坏疽。而且如果不能及时妥当地诊断和处理,有些症状会长期迁延,最终会演变为静脉血栓形成后综合征,长期影响患者的生活质量；还有一些病人可能会因血栓脱落而形成肺栓塞,直接危及病人生命,或者演变为慢性栓塞性肺动脉高压。目前,我国DVT的发病率尚无确切的统计资料,据国外资料报道,美国DVT年发病率约为千分之一。
个半世纪以前,Rudolph Virchow提出了血液瘀滞,血管壁的变化,和血液高凝状态是机体发生发展静脉血栓的主要机制,这个理论现今仍然适用,基本上所有的导致血栓的因素,都影响这三种机制之一。目前认为有很多导致个体易患静脉血栓的因素,可分为两类：一种是环境及后天生理病理因素的改变,如外伤,妊娠,围手术期,恶性肿瘤,机体制动,药物等影响机体的凝血机制；另一种是由遗传基因突变参与的凝血机制异常,有研究认为遗传基因突变在大于30%的个体参与到了深静脉血栓的发生过程。人们对各种基因多态性与血栓形成之间的关联性做了相当深入的研究,也发现了越来越多的基因多态现象确实参与到了凝血机制的改变和调节,并与静脉血栓构成相相关性。目前有研究认为亚甲基四氢叶酸还原酶C677T基因多态性由于会导致亚甲基四氢叶酸还原酶活性下降,进而影响同型半胱氨酸代谢,形成高同型半胱氨酸血症,从而促进血液高凝,增加机体血栓形成的风险；内皮细胞蛋白C受体A6936G基因多态性由于会提高血浆sEPCR的水平<进而增大SEPCR对蛋白C抗凝途径的负向调节,从而形血液高高凝,增加机体血栓形成的风险。对这两类基因多态现象的研究已经成为热点,本课题以临床病例为基础,采用一定的试验方法,对亚甲基四氢叶酸还原酶C677T基因多态性和因皮细胞蛋白C受体A6936G基因多态性与深静脉血栓形成的相关性进行了研究。
第一部分亚甲基四氢叶酸还原酶C677T基因多态性与深静脉血栓形成的相关性研究
亚里基四氢叶酸还原酶(MTHFR)在同型半胱氨酸(Hey)代谢中起着关键作用,可以催化5,10-亚甲基四氢叶酸转换为5甲基四氢叶酸,从而成为甲基的提供者参与维生素B12依赖的同型半胱氨酸向蛋氨酸转化；MTHFR降低,会导致Hey的代谢障碍,进而形高同型半胱氨酸血症。最近的研究已经证实高血浆Hey浓度是在深静脉血栓形成的勺危险因素之一，而MTHFR(C677T基因多态性已被报道可能会产生MTHFR酶的热不稳定性以及一定程度上降低该酶的活性,进而形成高同型半胱氨酸血症,从而导致机体易形成血栓。为了进一步深入研究探讨这一理论,我们设计了本项病例对照研究。
目的与方法
探讨血Hey水平及MTHFR C677T基因多态性与DVT发病的关系。在本课题的病例对照研究中,我们分析了DVT患者的MTHFR基因C677T多态性各基因型的频率，并与正常对照进行比较。此外,通过检测受试者血浆Hey水平，我们评估这一多态性与血浆Hey水平之间的关联,以及血浆Hey水平与DVT的关系。应用酶联关免疫吸附试验(ELISA)检测血浆Hey水平，采用限制旧性片段长度多态性聚合酶链反应(PCR RFLP)技术测定基因组DNA。
结果
MTHFR基因在677位置菜有3种基因型CC，CT和TT基因型，T为突变等位基因。在DVT组,T等位基因频率明显高于对照组。与CC基因型相比,TT基因型与DVT相关的风险显著增加(OR=3.200；95%CI,1.2937.922，P=0.010。与等位基因(相比,突变等位基因T与DVT的风险增加相关(OR=1.712;95%CI,1.095-2.675,P=0.018),DVT组的血浆Hey水平显著高于对照组而且MTHFR基因C677T突变对于血浆Hey水平有影响,突变等位基因T与血浆Hey水平升高有关。
结论
MTHFR677位点基因突变与个体发生DVT的风险增加之间存在关联。突变纯合子可以导致DVT、风险增加,突变等位基因T也与DVT风险增加有关。DVT患者有着比健康人群更高的血浆同型半胱氨酸水平,而且MTHFR基因C677T突变对于血浆Hcy水平有影响,突变等位基因T与血浆Hey水平升高有关。结论是,MTHFR C677T基因多态性可能是DVT发生的危险因素之一,而这种关联可能是通过血浆Hcy的浓度变化实现的。
第二部分内皮细胞蛋白C受体A6936G基因多态性与深静脉血栓形成的相关性研究
已知蛋白C (PC)系统是一种重要的抗凝血机制,在其辅酶蛋白S(PS)存在的条件下,活化蛋白C (aPC)使因子Va和Villa失活,从而减少凝血酶生成。内皮细胞蛋白C受体(EPCR),是一种在血管内皮细胞的表面的受体。这种受体可以与PC或aPC结合。这种结合可以提高PC的活化效率,加速aPC对因子Va和Villa灭活,从而实现抗凝。而可溶性内皮细胞蛋白C受体(sEPCR)在金属蛋白酶作用下从EPCR脱落,也可以与PC和aPC结合,但它与EPCR作用相反,而且产生了与EPCR的竞争性抑制,从而抑制了aPC的抗凝活性,导致高凝。因此血浆sEPCR水平被认为与机体的高凝倾向有关,sEPCR水平升高会导致机体易患血栓,而EPCR基因突变可能会产生sEPCR水平的升高。为了进一步探讨这个理论,我们对EPCR A6936G多态性、血浆sEPCR水平和DVT之间的关系进行了深入的研究。
目的与方法
本研究通过检测DVT病人以及正常对照人群EPCR A6936G多态性分布,并通过测定血浆sEPCR表达水平,探讨其在DVT形成中的作用。病例组由已确诊的DVT患者组成,对照组来自健康查体者。应用酶联免疫吸附试验(ELISA)检测血.浆sEPCR水平,采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术测定基因组DNA。
结果
DVT组突变等位基因G的频率显著高于对照组。相对于AA基因型,AG基因型(OR=2.502;95%CI,1.215-5.153),和等位基因G携带者AG+GG(OR=2.507;95%CI,1.238-5.076),都表现出增加了DVT的发生风险。突变等位基因G相对于等位基因A,也增加了DVT发生的风险(OR=2.178；95%C1,1.159-4.093)。DVT组血浆sEPCR水平显著高于对昭组,而突变等位基因G与血浆sEPCR水平升高有关。
结论
EPCR6936的基因突变与机体DVT发病的敏感性有关,突变等位基因G导致DVT发病危险增加。DVT患者具有比健康群体史更高的血浆sEPCR水平,而且血浆sEPCR水平与EPCR A6936G基因多态性存在关联,基因突变会导致血浆sEPCR水平升高。结论是,EPCR A6936G的基因多态性与血浆sEPCR水平升高有关,其可能是人群中发生DVT的危险因素之一。
课题创新性与意义
一.本研究结合了流行病学的研究方法和分子生物学的实验技术,首次同期联合检测了DVT患者和健康人群的MTHFR基因C677T和EPCR基因A6936G多态性的分布以及与其关联的功能蛋白Hey和sEPCR的表达水平,确定两个基因多态性与DVT之间的联系,以及基因多态性与功能蛋白表达水平的关系。为从病因学上在基因水平研究血栓性疾病增加了新的认识。
二.本课题体现了“基因-蛋白-疾病”的研究思路,由于本研究对象集中于特定的地理区域(山东),实验结果具有一定的地域代表性,同时实验结论也具有临床指导意义；此外,本研究从方法学上为进一步研究基因多态性提供了一定依据。
Backgrounds
Deep venous thrombosis (DVT) in medicine is a serious and common disease affecting all age groups. Its causes include a variety of pathological conditions and it often lead to hospitalization of patients, and severe conditions that require urgent intervention. About one and a half century ago, Rudolph Virchow proposed the main determinants that lead to DVT, summarizing that blood stasis, changes in the vessel wall, and blood hypercoagulability are the main mechanisms of the body for developing venous thrombosis. This theory is still applicable today and almost all of factors that lead to thrombosis, either systemie or of molecular level, have essentially affected one of the three mechanisms.There are a lot of mechanisms lead to the susceptibility to venous thrombosis clinically.which can be divided into two main categories:one category includes direct or indirect causes by trauma, systemic disease, or physiological state changes such as pregnancy, high-altitude, surgery, malignancy, body immobilization, and so on. The other is due to genetic mutations involving in the abnormal blood clotting mechanism. At present, due to the rapid progress of molecular genetics researches, and more accurate in vitro experiments, it has been found that in more than30%of individuals, genetic mutations participate in the mechanism of occurrence of DVT. Domestic research in this area is relatively rare. The present study is divided into two parts, respectively, related to endothelial protein C receptor A6936G gene polymorphism and methylenetetrahydrofolate reductase C677T gene polymorphism. Moreover, the relationship between the two polymorphisms and DVT were also studied.
Part1Deep Venous Thrombosis has Relationship with Methylenetetrahydropholate Reductase C677T Gene Polymorphism and Hyperhomocysteinemia
Methylenetetrahydrofolate reductase (MTHFR) plays a key role in homocysteine (Hey) metabolism,which can catalyze the reaction from5,10-methylenetetrahydrofolate to5-methylenetetrahydrofolate, thus becoming the methyl donor involved in the conversion from vitamin B12-dependent homocysteine to methionine. Reducing MTHFR concentration,it tend to be observed that thermal instability form of the enzyme, it will lead to high homocysteine concentration, which is characterized by mild to moderate increasing in plasma total homocysteine concentration. In vascular occlusive disease, the relationship with hyperhomocysteinemia has been reported. Recent studies have also demonstrated that high plasma total homocysteine concentration is a risk factor for deep venous thrombosis (DVT) in the general population.
MTHFR gene polymorphisms have been reported to be associated with high plasma homocysteine concentration. MTHFR gene C677T,can develop thermal instability and reduce the enzyme activity in vitro to a certain extent. C677T mutation in the homozygous individuals can moderately increase plasma homocysteine concentration, especially under the condition of low plasma folate level. Homozygotes of the C677T polymorphism in some studies have been regarded as a risk factor for venous thrombosis, however, not all studies support this conclusion. Opposite conclusions exist in some researches.Moreover, few domestic research has reported in this area.
Objective and Methods
To explore the relationship of blood I Icy levels and MTHFR C677T gene polymorphism and DVT incidence, in this case-control study, we analyzed the frequency of genotypes with MTHFR C677T polymorphism in DVT patients compared with normal individuals without DVT. In addition, we evaluated the association between this polymorphism and plasma Hey levels. PCR-RFLP was used to investigate the relationship between MTHFR C677T genotypes and DVT. The Hey levels were measured by enzyme-linked immunosorbent assay (ELISA) in DVT patients and control subjects.
Results
There are3genotypes,CC, CT and TT genotypes,in the MTHFR gene at position677in the two groups. The frequencies of T allele in DVT group was significantly higher than in the controls. The MTHFR C677T frequencies TT genotype was also significantly higher than in the controls. Compared with the CC genotype, the TT genotype was significantly correlated with a increased risk of DVT (OR=3.200;95%CI,1.293-7.922, P=0.010). Compared with the C allel, the T allel was significantly correlated with a increased risk of DVT(OR=1.712;95%CI,1.095-2.675, P=0.018).The plasma level of I Icy in the DVT group was significantly higher than that in the control group. And the level of Hcy in subjects with TT genotype was significantly higher than in subjects with CT genotype, meanwhile, the level of Hcy in subjects with CT genotype was significantly higher than in subjects with CC genotype.
Conclusion
The analysis in this study showed that the MTHFR677TT homozygote was a significant risk factor for DVT. And the I allel was correlated with a increased risk of DVT. There are differences in I Icy levels among the different genotypes impling that the mutation accounts for the changes of Hey levels. Hcy levels are elevated in DVT. In conclusion, our present observations allow us to suggest that the C677T mutation of the MTHFR gene, which is possible to cause hyperhomocysteinemia, is probably one of the candidate genetic risk factors of DVT.
Part2Endothelial cell protein C receptor gene6936A/G polymorphism is associated with deep venous thrombosis
Protein C (PC) system is an important natural anticoagulant mechanism. Under the condition of the existance of its coenzyme protein S, activated protein C (aPC) can make inactivation of factor Va and Ⅷa, thereby reducing thrombin generation. The activation of endothelial cell protein C receptor (EPCR), is a vascular endothelial cell surface receptor of newly discovered. This receptor combining with almost the same affinity with PC or aPC,can accelerate the activation of PC.Whereas, soluble endothelial cell protein C receptor (sEPCR) gets the effect of metalloproteinase, shedding from EPCR, being also combined with PC and aPC similar affinity. sEPCR is able to block the aPC with the combination of phospholipids and eliminate its effects of inactivation for factor Va, thereby inhibiting the anticoagulant activity of aPC. Several factors can influence level of sEPCR including gene polymorphisms. For further investigating the relation between EPCR A6936G polymorphism and DVT,we performed this case-control study. The relation between EPCR A6936G polymorphism and sEPCR level was also studied.
Objective and Methods
In this study, the aim of this study was to explore the role of gene mutation in the DVT formation. PCR-RFLP was used to investigate the relationship between EPCR A6936G polymorphism and DVT. The plasma soluble endothelial cell protein C receptor (sEPCR) levels were measured by enzyme-linked immunosorbent assay (ELISA) method in DVT patients and control subjects. All statistical analysis was performed for confirmation the possible relationships.
Results
Three genotypes, AA, AG and GG, in the EPCR gene at position6936were noted in the DVT group as well as in the control group. In the DVT group, the frequency of mutational genotypes was significantly higher than that in the control group (P<0.05). Furthermore, the frequency of the G allele in the DVT group was significantly higher than that in the control group (OR=2.178;95%CI,1.159-4.093). The plasma level of sEPCR in the DVT group was significantly higher than that in the control group. And the level of sEPCR in subjects with GG genotype was significantly higher than in subjects with AG genotype, meanwhile, the level of Hey in subjects with AG genotype was significantly higher than in subjects with AA genotype.
Conclusion
In this study, the results suggest an increased risk for venous thrombosis in EPCR6936mutational genotypes. The G allele indicates susceptibility to DVT and is associated with DVT pathogenesis. Moreover, our results indicate that the plasma sEPCR level is associated with the A6936G polymorphism of the EPCR gene. The plasma sEPCR level was elevated in DVT patients. Our findings suggest that the EPCR gene A6936G polymorphism may be a candidate risk factor for DVT.
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